The combination of nanomaterials and DNA has captured extensive attention in the fields of sensor, catalysis, biomedicine and material science. The applications of DNA functionalized MoS₂nanosheets (NSs) in biosensor and biomedicine in turn present an fascinating topic about biointerface science between DNA and MoS₂NSs. The surface interaction between DNA and MoS₂NSs was explored by adsorption and desorption experiments. The results indicated that van der waals force played an crucial role in DNA adsorption by MoS₂NSs. Additionally, it was proved that the quenching of dye labelled DNA probe by MoS₂NSs was attributed to fluorescence resonance energy transfer. Based on the extraordinarily high quenching efficiency of MoS₂NSs and different adsorption affinity of MoS₂NSs toward aptamer and aptamer-targeted protein, an aptameric biosensor was fabricated to detect carbohydrate antigen 15-3. The fabricated biosensor not only displayed high sensitivity with the detection limit of 0.0039 U/mL (S/N = 3), but also exhibited good stability and selectivity. The proposed sensing platform provided an easy-constructed and cost-effective protocol to detect other aptamer targeted biomolecules. The fundamental study of biointerface science on DNA and MoS₂NSs will facilitate their potential applications in various fields.